Genotyping technologies enable to construct high-resolution genetic maps for genomes of human and other organisms, resulting in LD blocks or r2 bins dependent of the method used. These LD blocks (haplotypes) can be used in association studies to localize genes responsible for a particular phenotype (e.g. disease). In other words, the aim of genotyping is to find correlations between genome organization (haplotype) and a particular phenotype.
It is becoming increasingly evident, that hundred thousands single nucleotide polymorphisms (SNP-s) have to be analyzed simultaneously to get a complete picture of haploblocks' (or r2 bin) arrangement. Thus, new methods are needed to accomplish this expansive and expensive task. Below is an overview of well-known genotyping methods that despite of their high throughput technology have also their limiting factors and therefore new cost-effective, flexible and focused genotyping methods are needed.
Genotyping and mutation detection is increasingly more substantial in clinical practice. Modern and reliable methods are alternative for RFLP, allele-specific PCR or sequencing in future. Flexible genotyping and mutation detection analyzes systems help to diagnose tens of candidate genetic markers in one reaction in short time as a routine procedure.                1. Detection based hybridization methods such as Affymetrix GeneChip (FIG. 1.) technology enables to analyze half a million SNP-s (Matsuzaki et al., 2004 1 & 2). Studied loci in genomic DNA are amplified without PCR with specific primers. An alternative way to reduce complexity of the genome is to use the restriction enzyme cut of genomic DNA, ligation of the fragments produced in this way to a universal linker and amplification with universal primers in PCR. Amplified regions are fragmented, labeled and hybridized to complementary oligonucleotides synthesized on microarray. In case of a perfect match, a duplex is formed and a signal from the perfect match pairings which is higher than signals from the mismatched pairing is detected. GeneChip microarray uses 25 base pairs (bp) long oligonucleotides containing a central nucleotide corresponding to the studied SNP. Hence, on both sides of a SNP, there are regions with 12 bp perfect match and in the middle, at the position of the SNP, a perfect match or a mismatch can occur.                    The key step of the method is DNA restriction and addition of universal linker sequences to the ends of all created fragments. Subsequently 250-2000 bp long fragments, containing the SNP(s) of interest are amplified with primers that anneal to the universal linker sequences (universal primer). With this approach a vast number of genomic sequences can be amplified with minimal cost. The limiting factor in this method is the number of restriction sites across the genome. Thus, SNP-s not located in the synthesized fragments (250-2000 bp), cannot be detected. Hence, this method in principle does not cover the whole genome and therefore the Illumina 300K array is as informative as Affymetrix 500K array in CEU population (Barrett and Cardon, 2006).                        2. Affymetrix Molecular Inversion Probe (MIP) method uses MIP molecules which are special “padlock” probes (Nilsson et al., 1994) for genotyping. MIP molecule is a linear oligonucleotide that contains specific regions, universal sequences, restriction sites and a Tag (index) sequence (16-22 bp). MIP hybridizes directly around the genetic marker/SNP of interest (FIG. 2).                    MIP method uses 1500 “padlock” probe sets that hybridize to genomic DNA in parallel (Hardenbol et al., 2003). In case of a perfect match, binding genomic homology regions are ligated by creating a circular molecule. After the first restriction, all molecules are amplified with universal primers. Amplicons are restricted again to ensure short fragments for hybridization on microarray. Generated short fragments are labeled and through Tag sequence hybridized to cTag (complementary strand for index) on array. After the formation of Tag-cTag duplex, a signal is detected.            Despite the complexity of experimental procedures, it is possible to amplify up to 10 000 SNP-containing sequences in one reaction. Thereby polymorphisms can theoretically be detected in any genomic region of interest (Hardenbol et al., 2005). There are two MIP probes for each allele, thus the method uses four probes (70 to 100 bp), a universal primer and a cTag sequence on array for a SNP detection in both strand.                        3. Illumina GoldenGate genotyping platform outstands primarily with an original solution of the gene chip, but the molecular approach amplifying genomic regions is similar to MIP probes. Genomic DNA fragments are attached to specific particles, followed by hybridization with specific probe molecules (FIG. 3). Probe molecules are supplied with three different universal sequences and a Tag sequence, situated between the specific region and universal primer (Gunderson et al., 2005). Specific primers are hybridized to genomic DNA and designed to be allele specific, meaning that in order to identify a SNP, an oligonucleotide must be synthesized in a way that its 3′ end binds to the SNP under study.                    Hence, to determine a SNP two probe molecules, each supplied with a different universal primer sequence, are needed. In case of a 3′ perfect base-pairing at the 3′-end one primer is elongated by primer extension reaction up to the other oligonucleotide, followed thereafter by ligation and generation of a linear molecule. The formed molecule includes two universal primer binding sites and a Tag sequence.            The formation of a linear molecule enables the PCR amplification with universal primers which in turn are supplied with two different fluorescence labels to detect homo- or heterozygosity at the studied position. GoldenGate method uses hybridization-based signal detection through formation of Tag-cTag complex.                        4. The Infinium 1 method of Illumina (FIG. 4) enables similarly to Affymetrix GeneChip technology a genome-wide analysis and to test up to half a million SNPs on a gene chip so far. The method does not use PCR to amplify the studied loci. Instead, genomic DNA is amplified using the WGA method (Whole Genome Amplification) (Gunderson, Steemers et al., 2005).                    Amplified genomic DNA is fragmented and hybridized to oligonucleotides (75 bp) on the gene chip. After hybridization from 16 to 18 hours, unbound or mismatched fragments are removed during a specific wash step.            The studied genotype on the gene chip is determined by using allele-specific oligonucleotides (Infinium1), where the 3′-terminal nucleotide is complementary to the SNP and which are primer extended away from the marker. In case of a perfect match primer sequence is elongated by DNA polymerase with fluorescence-labeled desoxynucleotides, which in case of the switch in the chain give a fluorescence signal. In order to detect a SNP two oligonucleotides are used on a gene chip.            Infinium 2 platform incorporates two-color single base extension to detect a single nucleotide polymorphism with 100K BeadChip, using only one oligonucleotide per SNP (Steemers et al., 2006).            Compared to GeneChip technology Infinium 1 and 2 are not defined by the restriction sites in genomic DNA and therefore has a good potential to solve the genotyping of all SNPs.                        